Estrogen is a hormone secreted in major quantities by the ovaries. The principal function of estrogens is to cause cellular proliferation and growth of the tissues of the sexual organs and of other tissues related to reproduction. Estrogens exert their effects primarily on the fallopian tubes, breasts, skeleton, protein deposition, metabolism, fat deposition, hair distribution, skin, and electrolyte balance. While the exact mechanism has yet to be identified, estrogens have been known for many years to also play a role in the proliferation of certain types of cancer.
Three estrogens are present in significant quantities in the plasma of females: 17.beta.-estradiol, estrone, and estriol which have the following formulas: ##STR1##
The principal estrogen secreted by the ovaries is 17.beta.-estradiol. Small amounts of estrone are also secreted, but most of this is formed in the peripheral tissues from androgens secreted by the adrenal cortices and by the ovarian thecal and stromal cells. Estriol is an oxidative product derived from both estradiol and estrone, the conversion occurring mainly in the liver.
Estrogen degradation takes place in the liver wherein the liver conjugates the estrogens to form glucuronides and sulfates, and about one-fifth of these conjugated products are excreted in the bile while most of the remainder are excreted in the urine. Also, the liver converts the potent estrogens, estradiol and estrone, into the almost totally inactive estrogen estriol.
In mammalian cells, the estrogens 17.beta.-estradiol (E.sub.2) and estrone (E.sub.1), which are continuously interconverted by 17.beta.-oxidoreductase, are generally metabolized via two major pathways: hydroxylation at the 16.alpha.-position or hydroxylation at the 2- or 4-position by cytochrome P-450. The latter pathway produces catechol estrogens (CE).
CE are typically conjugated by catechol-O-methyltransferases to give their monomethoxy derivatives. These enzymes are protective, because only non-methylated CE can be oxidized to their quinones (CE-Q) by peroxidases and cytochrome P-450.
Once CE form quinones, they can be acted upon by glutathione to form estrogen-glutathione adducts. These adducts in turn are broken down in the kidneys to produce cysteine and N-acetylcysteine adducts. The cysteine/N-acetylcysteine adducts are then secreted in the urine.
The role of estrogens in the induction of cancer has generally been related to stimulation of proliferation by receptor-mediated processes. Nandi, S. et al. (1995), "Hormones and Mammary Carcinogenesis in Mice, Rats, and Humans: A Unifying Hypothesis." Proc. Natl. Acad. Sci. USA, 92, 3650-3657. Estrogens can also play another important role by generating electrophilic species that can covalently bind to DNA to initiate cancer. Liehr J. G. (1990), "Genotoxic Effects of Estrogen." Mutat. Res. 238, 269-276. The present inventors have found evidence that CE-Q are the ultimate carcinogenic forms of estrogens, since these electrophiles can covalently bind to nucleophilic groups on DNA via a Michael addition. Furthermore, redox cycling generated by reduction of CE-Q to semiquinones and subsequent oxidation back to CE-Q can generate hydroxyl radicals that can cause additional DNA damage as proposed by Liehr et al. and Nutter et al. Liehr, J. G. et al. (1986) "Cytochrome P-450-Mediated Redox Cycling of Estrogen". J. Biol. Chem. 261, 16865-16870. Mutter, L. M., et al. (1994) "An o-Quinone Form of Estrogen Produces Free Radicals in Human Breast Cancer Cells: Correlation With DNA Damage." Chem. Res. Toxicol. 7, 23-28.
Several lines of evidence suggest that the 4-hydroxyCE are critical intermediates in the pathway leading to estrogen-induced cancer. Malignant renal tumors are induced in Syrian golden hamsters by treatment with E.sub.1 or E.sub.2, suggesting that E.sub.1 or E.sub.2 can be procarcinogenic compounds. Li, J. J. et al., (1983) "Relative Carcinogenic Activity of Various Synthetic and Natural Estrogens in the Syrian Hamster Kidney". Cancer Res. 43, 5200-5204. The CE 4-hydroxyestrone (4-OHE.sub.1) and 4-hydroxyestradiol (4-OHE.sub.2) also induce renal tumors in hamsters, whereas the corresponding 2-OH isomers do not. Liehr, J. G. et al., (1986), "Carcinogenicity of Catechol Estrogens in Syrian Hamsters", J. Steroid. Biochem. 24, 353-356. Furthermore, an estrogen-4-hydroxylase activity has been identified not only in hamster kidneys, but also in other organs prone to estrogen induced cancer, such as rat pituitary, mouse uterus, human MCF-7 breast cancer cells, human uterine myometrial tumors and human breast cancer tissues. Ricci, M. J., et al., (1995), "Predominant 4-Hydroxylation of Estradiol by Microsomes of Neoplastic Human Breast Tissue", Proc. Amer. Assoc. Cancer Res. 36, 255.
Adducts formed by direct reaction of CE-Q with DNA have been compared to those formed after activation of CE by horseradish peroxidase. Dwivedy, I. et al., (1992), "Synthesis and Characterization of Estrogen 2,3- and 3,4-quinones. Comparison of DNA Adducts Formed by the Quinones Versus Horseradish Peroxidase-Activated Catechol Estrogens". Chem. Res. Toxicol. 5, 828-833. In these studies, however, the structures of adducts were not identified.
The inventors have further evidence that the detection of N-acetylcysteine-estrogen and cysteine-estrogen adducts are also useful biomarkers for determining an individual's susceptibility to estrogen-related forms of cancer. Further, reactions of CE-Q with nucleosides and cysteine would provide authentic adducts and valuable insight into the mechanism of their formation. By studying the formation of these adducts, the mechanism behind estrogen-induced cancer would be more easily identified, thereby providing a means of diagnosing people at risk for estrogen-induced cancer, as well as aiding in the detection and treatment of estrogen-induced cancer. In addition, the synthetic adducts would serve as standard compounds for elucidating adducts formed by reaction of estrogen metabolites with DNA and glutathione.
It is therefore an object of the present invention to provide synthetic estrogen-purine base and estrogen-mercapturate adducts.
It is a further object of the present invention to provide diagnostic methods for detecting the presence of estrogen-purine base and estrogen-mercapturate adducts.
It is a further object of the present invention to provide fluorescent derivatives of the estrogen-purine base and estrogen-mercapturate adducts for use in fluorescent detection of those adducts.
It is still a further object of the present invention to provide a chemical test for detecting the presence of estrogen-purine base and estrogen-mercapturate adducts in body fluid samples.
It is yet another object of the present invention to provide an immunoaffinity matrix for purifying estrogen-purine base and estrogen-mercapturate adducts from body fluid samples for analysis by high performance liquid chromatography.
A further object of the present invention is to provide specific antibodies and capture agents for use in immunoassays for naturally occurring estrogen-purine base and estrogen-mercapturate adducts.
It is yet another object of the present invention to provide a method for detecting estrogen-induced cancer by assaying for the presence of estrogen-purine base and estrogen-mercapturate adducts in a body fluid sample.
It is yet another object of the present invention to provide synthetic antigens comprising estrogen-purine base and estrogen-mercapturate adducts.
It is still another object of the present invention to provide monoclonal antibodies which recognize the synthetic antigens comprising estrogen-purine base and estrogen-mercapturate adducts.
These and other objects of this invention are described more fully hereinafter.